In the development of colorectal cancer (CRC), tumorsuppressor genes such as APC, p53, and genes on chromosome 18q (DCC, SMAD2, and DPC4/SMAD4) are inactivated by mutations or by chromosomal deletions (1, 2). Some CRCs have microsatellite instability caused by inactivation of mismatch repair genes such as hMSH2 or hMLH1 (3). In addition, epigenetic inactivation by hypermethylation of promoter regions of various tumor suppressor genes such as p16, APC, VHL, and hMLH1 have been found in CRCs (4-10) and used as molecular markers of CRC (11, 12).
Methylation of cytosines in CpG islands in the promoter region affects promoter activity and can down-regulate gene transcription (5). Because the promoter hypermethylation of genes in cancer cells is as significant as deletions or mutations (13-15), hypermethylation of key regulatory genes can play a significant role in transformation and tumor progression. Progression of transformed cells requires regulatory gene inactivation that promotes growth, dedifferentiation, invasion, and/or metastasis.
Transcription factors containing a basic helix-loop-helix (bHLH) motif regulate the expression of certain tissue-specific genes (16) and have important roles in cell differentiation and embryonic developmental processes. DNA-binding activity of the bHLH proteins is dependent on formation of homo- and/or heterodimers. ID family proteins, which are distinct members of the helix-loop-helix (HLH) protein family, contain the HLH-dimerization domain but lack the DNA-binding basic domain. Consequently, ID proteins dominantly inhibit binding to DNA and transcriptional transactivation by forming heterodimers with bHLH proteins and modulate various key developmental processes (17). Currently, four known human ID proteins have been identified. Expression studies have shown that ID proteins play critical roles in early embryonic development (18-20). They are also involved in angiogenesis, lymphocyte development, cell cycle control, and cellular senescence (21-23). The involvement of ID proteins in neoplastic processes has been suggested. Increased ID1 and ID2 expression has been reported in various tumor types, including adenocarcinomas arising from the colon and pancreas (24, 25). Transgene expression of ID1 and ID2 in mice has resulted in tumor formation in the intestinal epithelium and lymphoid organs, respectively (26, 27). Expression of ID3 has been more variable; studies report both up-regulation (24, 28) and down-regulation (29, 30) in different tumor types.
ID4 gene has a relation with growth and differentiation of cells, as reported with oligodendrocytes (31). Recently, it was reported that ID4 promoter is hypermethylated in 30% of primary gastric cancers, and expression is down-regulated in most gastric cancer cell lines by hypermethylation of the promoter region (32). Despite the structural similarity, ID4 is known to have some differences from the other three known ID gene members. Unlike ID1, ID2, and ID3, the immunoreactivity of which is significantly elevated in CRCs compared with normal epithelium (24), ID4 has a more restricted expression pattern during murine and avian embryogenesis and is expressed at more advanced stages of differentiation in tissues (18-20). In the development of murine stomach, ID4 expression is restricted to the ventral part where cells grow slower, whereas other ID members are expressed in the dorsal part of the stomach where cells proliferate faster (18). Information about ID4 function, expression, and regulation of tumor progression is very limited, and there are no published studies of ID4 in CRC to date.
The presence of axillary lymph node metastases is the most significant prognostic factor for patients with breast cancer (Fisher et al., 1993; Fitzgibbons et al., 2000). For patients with early breast cancers, the ability to predict nodal metastasis could alleviate the need for axillary staging. The rate of nodal metastasis in T1 (≧2.0 cm) breast cancers is reportedly from 18% to 31% (Barth et al., 1997; Carter et al., 1989; Holland et al., 1996). There are several known risk factors of lymph node metastasis for invasive breast cancer to date. For nodal metastasis of T1 breast cancer, lymphovascular invasion, tumor size, histologic grade, histologic type, and estrogen receptor (ER) status are the major risk factors of pathological findings (Brenin et al., 2001).
Advanced breast cancers reportedly express ID1 (Lin et al., 2000; Singh et al., 2002), and ID1 expression is associated with poor clinical outcome of patients with node-negative breast cancer (Schoppmann et al., 2003). By contrast, expression of ID2 is low in invasive breast cancer and correlates with noninvasive phenotype (Itahana et al., 2003). Expression of ID3 or ID4 in breast cancer has not been well studied. In breast cancer, animal studies have demonstrated that ID4 regulates mammary epithelial cell growth and differentiation, and overexpression of this protein in rat mammary gland carcinomas correlates with proliferation, invasiveness, and tumor weight (Shan et al., 2003). However, certain human primary breast cancers reportedly have low or no expression of ID4 protein by immunohistochemistry (IHC) (Welcsh et al., 2002), suggesting downregulation of the ID4 gene.